Organization of the mouse Ruk locus and expression of isoforms in mouse tissues

The University of Edinburgh, Edinburgh, Scotland, United Kingdom
Gene (Impact Factor: 2.14). 08/2002; 295(1):13-17. DOI: 10.1016/S0378-1119(02)00821-1
Source: PubMed


Ruk is a recently identified gene with a complex pattern of expression in mammalian cells and tissues. Multiple Ruk transcripts and several protein isoforms have been detected in various types of cells. Ruk proteins have multidomain organization characteristic of adapter proteins involved in regulation of signal transduction. Interaction of some Ruk isoforms with several signalling proteins, including the p85 regulatory subunit of the Class IA PI 3-kinase, c-Cbl and Grb2, has been demonstrated. Ruk(l), an isoform with three SH3 domains, inhibits lipid kinase activity of the PI 3-kinase in vitro; overexpression of this protein induces apoptotic cell death of primary neurons in culture and changes in membrane trafficking in other cultured cells. However, shorter isoforms of Ruk block pro-apoptotic effect of Ruk(l), suggesting that expression of different combinations of Ruk proteins in cells could be involved in the regulation of their survival and other intracellular processes. To understand the mechanism of differential expression of Ruk proteins we studied organization of the mouse Ruk gene and its transcripts. Twenty-four exons of the Ruk gene span over 320 kb of the mouse chromosome X. Analysis of cDNA clones, ESTs and products of RT-PCR amplifications with different combinations of primers revealed how alternative splicing and promoter usage generate a variety of Ruk transcripts and encoded protein isoforms in different mouse tissues.

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    • "Previously described protocols were used for Western blotting (Buchman et al., 2002). Protein levels were quantified using Cy3-or Cy5-conjugated secondary antibodies (Invitrogen, CA) and FluorChem Q MultiImage III system (Cell Biosciences, CA). "
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    • "Proteins were resolved in SDS-PAGE and transferred to Hybond P membrane (GE Healthcare, Little Chalfont, UK). Membranes were incubated with primary rabbit polyclonal anti-FLAG (Sigma, USA), anti-Nedd4 (Abcam, UK), anti-Nedd4-2 [30], or anti-neomycin phosphotransferase II (Millipore, USA), mouse monoclonal anti-α-tubulin, clone DM1A antibodies (Sigma, USA) or affinity purified rat anti-MAK-V antibodies [8] followed by secondary anti-rabbit, anti-mouse or anti-rat horseradish peroxidase-conjugated antibodies (HRP; GE Healthcare, USA) as described previously [31]. To detect GST, anti-GST HPR conjugate (GE Healthcare, USA) was used. "
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